Nucleic acids encoding for antifungal bifunctional molecules for treating fungal infection

ABSTRACT

The present invention is directed to nucleic acids encoding for fusion peptides comprising a fungal targeting agent and a channel-forming domain consisting essentially of amino acids 451-626 of colicin Ia, as well as vectors having the nucleic acids of the invention and host cells having the vectors. The fusion peptides of the peptides of the present invention are particularly useful for the treatment of fungal infections in a wide variety of organisms. The fusion peptides can be prepared from the nucleic acids, such as when a vector having the nucleic acid is included in a host cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationhaving U.S. Ser. No. 11/338,117, filed in the U.S. on Jan. 23, 2006 andpublished as Pre-Grant Publication 2006/0264370now U.S. Pat. No.7,915,382, which application claims priority under 35 U.S.C. 119(a) toChinese Patent Application No. 2005100202199, filed on Jan. 21, 2005,which applications are both incorporated herein by specific reference intheir entirety.

BACKGROUND

Fungus is opportunistic pathogens in humans. Fungus typically does notinfect healthy tissues, yet once tissue defense mechanisms have beencompromised, they can readily infect the tissue. One typical model ofthis opportunistic fungal infection is candidiasis, which is caused byCandida albicans.

Candida albicans occurs as normal flora in the oral cavity, genitalia,large intestine, and skin of approximately 20% of humans. The risk ofinfection increases in children and pregnant women; people who usecertain antibiotics or have nutritional and organic disease orimmunodeficiency (e.g., AIDS) or trauma; and people with invasivedevices, e.g., pacemakers. Candida albicans and its close relativesaccount for nearly 80% of nosocomial fungal infections and 30% of deathsfrom nosocomial infections in general.

Historically, opportunistic fungal infections in hospitalized patientswere rather unusual. Textbooks from the past described these agents ascommon contaminants with weak pathogenic potential, and infections wereconsidered extreme deviation from the normal. Older ideas concerningthese so-called harmless contaminants are now challenged because inthose days immunodeficient and debilitated patients had died from theirafflictions long before fungal infection took place. However, currently,with the advent of innovative surgeries, drugs, and other therapies thatmaintain such patients for expected periods, the survival rates ofpatients have significantly increased and the number of compromisedpatients has thus increased. One clinical dilemma that cannot becompletely eliminated, even with rigorous disinfections, is the exposureof such patients to potential fungal pathogens from even normal flora.Fungal infections in such high-risk patients progress rapidly and aredifficult to diagnose and treat. In one study, fungi causedapproximately 40% of the deaths from clinically acquired infections. Upto 5% of all nosocomial opportunistic fungi cause infections.

Fungi also present special problems in chemotherapy. A majority ofchemotherapeutic drugs used in treating bacterial infection aregenerally ineffective in combating fungal infection. Moreover, thesimilarity between fungal and human cells often means drug toxic tofungal cells are capable of harming human cells. A few drugs withspecial antifungal properties have been developed for treatment ofsystemic and superficial fungal infections. For example, macrolidepolyenes represented by amphotericin B, have a structure that mimicssome cell membrane lipids. Amphotericin B which is isolated from aspecies of streptomycin is by far the most versatile and effective ofall antifungal drugs. The azoles are broad-spectrum antifungal drugswith a complex ringed structure. As one of the most effective azoledrugs, fluconazole, is used in patients with AIDS-related mycoses.

Magnaporthe grisea is the pathogen of a devastating fungal disease ofrice plants known as rice blast. The fungus can also cause a similardisease in over 50 grasses, including economically important crops suchas barley, wheat, and millet. Fusarium is another important genus offungal pathogens, responsible for devastating diseases such as cerealscab.

SUMMARY

The present invention is directed to novel nucleic acid moleculesencoding for peptides that include a fungi specific targeting agent,e.g., a pathogenic fungal peptide pheromone, and a channel-formingcolicin or a channel-forming fragment thereof (also referred to hereinas “domain”). Peptides comprising a pheromone as the fungi specifictargeting agent, and a colicin domain, are referred to herein as“pheromonicin peptides”.

The molecular structure of the formed peptides may have the C-terminusof colicin or a channel-forming domain linked with the N-terminus of afungi specific targeting agent, e.g., a fungal pheromone, or theN-terminus of colicin may be linked with the C-terminus of a fungispecific targeting agent e.g., a fungal pheromone. The fungal pheromonecan be from a pathogenic fungus, e.g., Candidas. The molecular weight ofthe peptide may vary, e.g., from about 26,000 to about 70,000 daltons.

The nucleic acid molecules of the present invention may be formed by avariety of methods. One method of forming a peptide of the presentinvention is by inserting a nucleic acid molecule encoding a fungalpheromone into a selected position of a nucleic acid molecule encoding acolicin, or a channel forming domain thereof, then transfecting themutant plasmid into a host cell, e.g., E. coli, to produce the peptide.

In one embodiment, the peptides encoded by the nucleic acid molecules ofthe present invention are useful in treating infections of Candidas orAspergillus or Magnaporthes or Fusarium. Exemplary infections are thosecreated by Candida albicans, Candida tropicalis, Candida parapsilokis,Candida krusei, Candida dubliniensis, Cryptococcus neoformans, A.fumigatus, A. flavus, A. niger, Magnaporthe grisea and Fusariummoniforme.

The invention further provides vectors having the nucleic acid moleculesthat encode the peptides of the invention. The invention also providescells, e.g., host cells, comprising the vectors of the invention.

Host cells, including bacterial cells such as E. coli, insect cells,yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) orCOS cells), can be used to produce the peptides of the invention. Othersuitable host cells are known to those skilled in the art. The inventionthus provides methods for producing the peptides of the inventioncomprising the steps of culturing the host cells of the invention andisolating the peptides of the invention therefrom.

In another embodiment, the invention provides a method for preparing apeptide which inhibits growth of a fungus comprising: (i) inserting anucleic acid molecule encoding colicin, or a channel forming domainthereof, into a selected position of a nucleic acid molecule encoding afungal targeting agent, e.g., a pheromone; (ii) transfecting the mutantplasmid into a host cell, e.g., an E. coli cell; and (iii) allowing saidhost cell to produce said peptide. In further embodiments, the peptidemay be purified from the cells.

In another embodiment, the invention provides a method for preparing afusion peptide comprising: (i) incorporating a nucleic acid moleculeencoding the peptide chain of colicin Ia with a nucleic acid moleculeencoding a fungal pheromone such as Candida albicans alpha-matingpheromone; and (ii) introducing said nucleic acid molecule encoding thepeptide chain of colicin Ia incorporated with said fungal pheromone afollowing the C-terminus of the colicin Ia to form a nucleic acidmolecule that encodes a 639 residue peptide.

In another embodiment, the invention provides a method for preparing afusion peptide comprising: (i) incorporating a nucleic acid moleculeencoding a peptide chain of colicin Ia with a nucleic acid moleculeencoding a fungal pheromone such as Candida albicans alpha-matingpheromone; and (ii) introducing said nucleic acid molecule before theN-terminus of said colicin Ia to form a nucleic acid molecule thatencodes a 639 residue peptide.

In one embodiment, the invention provides a method of treating a subjecthaving a fungal infection comprising: administering to a subject atherapeutically effective amount of a fusion peptide of the presentinvention, e.g., a peptide comprising a colicin Ia with a fungaltargeting agent, e.g., a pheromone. Said subject may have a Candidas orAspergillus or Magnaporthe or Fusarium infection. Specifically, Candidasor Aspergillus or Magnaporthe or Fusarium may be selected from the groupconsisting of Candida albicans, Candida tropicalis, Candidaparapsilokis, Candida krusei, Candida dubliniensis, Cryptococcusneoformans, A. fumigatus, A. flavus, A. niger, Magnaporthe grisea andFusarium moniforme. The peptides of the instant invention can also beused to treat clinical fugal infections and other fungal infections incrops.

The peptides of the invention can be incorporated into pharmaceuticalcompositions suitable for administration to a subject. In a preferredembodiment, the pharmaceutical composition comprises a peptide of theinvention comprising the C. albicans alpha-pheromone and apharmaceutically acceptable carrier.

The term, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous or parenteral administration(e.g., by injection). Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of acids and other natural conditions, which may inactivatethe compound.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and following information as well as other features ofthis disclosure will become more fully apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings. Understanding that these drawings depict onlyseveral embodiments in accordance with the disclosure and are,therefore, not to be considered limiting of its scope, the disclosurewill be described with additional specificity and detail through use ofthe accompanying drawings, in which:

FIG. 1 schematically depicts the structure of a recombinant plasmid thatcontains the gene of colicin Ia with the gene of Candida albicansalpha-mating pheromone inserted following the C-terminus of colicin Iain the plasmid pET-15b to form a plasmid referred to herein as pCHCCA1.

FIG. 2 schematically depicts the structure of a recombinant plasmid thatcontains the gene of colicin Ia with the gene of Candida albicansalpha-mating pheromone inserted following the N-terminus of colicin Iain the plasmid pET-15b to form the plasmid referred to herein aspCHCCA2.

FIG. 3 depicts a growth inhibition assay wherein ATCC 10231 C. albicanscells were grown in M-H solid medium and exposed to (A) borate stocksolution as control, (B) 50 ul amphotericin B (1 ug/ml), (C) 50 ulfluconazole (3 ug/ml), (D) 50 ul pheromonicin-SA (Ph-SA)(50 ug/ml), thefusion peptide against Staphylococcus aureus and (E) 50 ulpheromonicin-CA1 (Ph-CA1)(50 u/ml), the peptide produced by the pCHCCA1plasmid.

FIG. 4 depicts the inhibition effects of Ph-CA against the growth ofATCC 10231 C. albicans cells in M-H liquid medium. The amount ofsedimentary fungal filaments at the bottom of flasks indicated theinhibition effects of treatment agents. (A) Control, (B) fluconazole,(C) amphotericin B, (D) Ph-SA, (E) Ph-CA2 produced by pCHCCA2 plasmidand (F) Ph-CA1.

FIG. 5 depicts the inhibition effects of Ph-CA against the growth of C.albicans cells in liquid medium. The amount of spores and filaments ofATCC 10231 C. albicans cells indicated the inhibition effects. (A)Control, (B) fluconazole, (C) amphotericin B, (D) Ph-SA, (E) Ph-CA2 (F)Ph-CA1. X400.

FIG. 6 depicts the fluorescent imaging of ATCC 10231 C. albicans cellstreated by Ph-CA1 and stained with 50 nM FITC/600 nM propidium iodide.(A) Control, cells were stained by FITC as green, (B) cells became redafter 24 hrs Ph-CA1 treatment (10 ug/ml). X400.

FIG. 7 depicts a growth inhibition assay wherein Huaxi 30168Cryptococcus neoformans cells were grown in M-H solid medium and exposedto (A) and (B) 100 ul amphotericin B (2 ug and 0.5 ug/ml respectively),(C) and (D) 100 ul Ph-CA1 (50 ug and 25 ug/ml respectively).

FIG. 8 depicts a growth inhibition assay wherein Huaxi 30255 Aspergillusflavus cells were grown in PDA solid medium and exposed to (A), (B) and(C) 100 ul tricyclazole (5 mg, 0.5 mg, and 0.05 mg/ml respectively), (D)100 ul Ph-CA1 (50 ug/ml).

FIG. 9 depicts a growth inhibition assay wherein ACCC 30320 Magnaporthegrisea cells were grown in PDA solid medium and exposed to (A) 100 ulamphotericin B (0.5 ug/ml), (B) 100 ul tricyclazole (0.5 mg/ml), (C) and(D) 100 ul Ph-CA1 (25 ug and 50 ug/ml respectively).

FIG. 10 depicts a growth inhibition assay wherein ACCC 30133 Fusariummoniforme cells were grown in PDA solid medium and exposed to (A)control, (B) 100 ul amphotericin B (0.5 ug/ml), (C) Ph-CA1 100 ul Ph-CA1(50 ug/ml) and (D) 100 ul tricyclazole (0.5 mg/ml).

FIG. 11 depicts in vivo activity of Ph-CA1 against systemic candidiasis.The C. albicans infected mice were untreated or treated byintraperitoneal amphotericin B or Ph-CA1.

FIG. 12 depicts in vivo activity of Ph-CA1 against systemic candidiasis.The C. albicans infected mice were untreated or treated by intravenousamphotericin B or Ph-CA1.

FIG. 13 depicts the microscopic view of visceral organs of mice treatedwith Ph-CA1 30 days. (A) liver, (B) kidney and (C) spleen stained withhematoxylin and eosin. 100X.

All arranged in accordance with at least one of the embodimentsdescribed herein, and which arrangement may be modified in accordancewith the disclosure provided herein by one of ordinary skill in the art.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

The antifungal peptides of the present invention comprise a fungispecific targeting agent e.g., a fungal pheromone, and one or morechannel-forming colicins or channel-forming domains thereof. Themolecular structure is generally either the C-terminus of a colicin orchannel-forming domain thereof, linked with the N-terminus of the fungalspecific targeting agent, or the N-terminus of the colicin or channelforming domain thereof, linked with the C-terminus of a fungal specifictargeting agent. Although full-length colicin may be used in the methodsand compositions of the invention, in some embodiments, only achannel-forming domain is used. In a preferred embodiment, the colicinchannel-forming domain consists essentially of amino acids 451-626 ofcolicin Ia.

Colicins are protein toxins produced by strains of E. coli. They aregenerally classified into groups corresponding to the outer membranereceptor on sensitive E. coli cells to which they bind, with colicinsthat bind to the BtuB protein, the high affinity receptor for vitaminB12, being known as the E group. E-type colicins are about 60 kDaproteins that have three functional domains each implicated in one ofthe three stages of cell killing. The C-terminal domain carries thecytotoxic activity, the central domain carries the receptor-bindingactivity, and the N-terminal domain mediates translocation of thecytotoxic domain across the outer membrane. Three cytotoxic activitiesare found amongst E-type colicins: (i) a pore-forming ion channel thatdepolarizes, the inner membrane (colicin E1); (ii) an H—N—H endonucleaseactivity that degrades chromosomal DNA (colicins E2, E7, E8 and E9); and(iii) ribonuclease activities (colicin E3, E4, E5 and E6).Colicin-producing bacteria are resistant against the action of their owncolicin through possession of a small immunity protein that inactivatesthe cytotoxic domain. After binding to E. coli cell surface receptors,E-type colicins are translocated to their site of action by a toldependent translocation system.

The peptides of the present invention maybe prepared by inserting anucleic acid molecule encoding a fungal pheromone into the selectedposition of a nucleic acid molecule encoding a colicin, or a channelforming fragment thereof. The resulting transfected mutant plasmid maythen inserted into a host cell, e.g., E. coli, to produce the peptide.Colicin Ia has the nucleic acid sequence set forth in SEQ ID NO: 1.Candida albicans alpha-mating pheromone has the nucleic acid sequenceset forth in SEQ ID NO:2 and the amino acid sequence set forth in SEQ IDNO:3.

The peptides of the invention may be used to treat subjects having afungal infection, e.g., Candidas, Cryptococcus, Aspergillus,Magnaporthes or Fusariums. Exemplary fungal infections are oral thrush,oesophageal thrush (Oesophagitis), cutaneous (skin) candidiasis, vaginalyeast infection or candida vaginitis, balanitis, and systemiccandidiasis. The peptides of the invention may also be used to treatdevastating fungal infections in crops.

EXAMPLES Example 1

A fusion peptide that has been identified as pheromonicin-CA1 (Ph-CA1)was created incorporating a peptide chain of colicin Ia with a Candidaalbicans alpha-mating pheromone, wherein the pheromone was c-terminal tothe colicin Ia to produce a polynucleotide having the nucleic acidsequence of SEQ ID NO:4 which encodes a polypeptide having the aminoacid sequence of SEQ ID NO:5.

Example 2

A second fusion peptide denominated as pheromonicin-CA2 (Ph-CA2) wascreated by incorporating a peptide chain of colicin Ia with a Candidaalbicans alpha-mating pheromone, wherein the pheromone is n-terminal tothe colicin Ia, to produce a polynucleotide having nucleic acid sequenceof SEQ ID NO:6 which encodes a polypeptide having the amino acidsequence of SEQ ID NO:7.

Results

Ph-CA1 had definite antifungal effect on Candida albicans (ATCC 10231)in vitro and in vivo. In contrast, Ph-CA2 almost had no effect. One invitro cell growth inhibition assay was performed with M-H or PDA solidmediums. About 5 ul Cells (10⁸ CFU/ml) of Candida albicans (ATCC 10231),Cryptococcus neoformans (Huaxi 30168 strain, clinical isolated strain byWest China Hospital, Sichuan University), Aspergillus flavus (Huaxi30255 strain), Magnaporthe grisea (ACCC 30320 strain, SpeciesConservation Center, Chinese Academy of Agriculture Sciences), orFusarium moniforme (ACCC 30133 strain) were inoculated on the surface of10 ml M-H or PDA solid mediums contained in disks. Then 50-100 ulamphotericin B (0.5 ug to 2 ug/ml) or fluconazole (3 ug/ml) ortricyclazole (0.05 mg to 5 mg/ml) or Ph-CA1 (25 to 50 ug/ml) eitherrinsed in a piece of filter paper or contained in a container then beingplaced on the surface of the medium, and incubated at 35° C. for 2 to 4days.

As shown in FIG. 3, only an inhibition-zone surrounds Ph-CA1, while nosimilar zones were observed with other agents. FIGS. 7 to 10 show thatPh-CA1 had definite antifungal effects against correspondingCryptococcus neoformans, Aspergillus flavus, Magnaporthe grisea andFusarium moniforme cells. On a molar basis, such antifungal effects wereone hundred to one thousand times greater than that of known antifungalantibiotics.

In vitro cell growth inhibition assays were performed in 100 ml Klettflasks containing 10 ml of M-H medium which were monitoredturbimetrically with a BioRad 550 microplate reader at OD595 nm every 60min. The filament (mycelium) precipitation at the bottom of flask wascounted with a digital photo-recorder every 6 hrs. Cells were inoculatedto an initial cell density of about 2.5×10⁵ CFU/ml and shaken at 200 rpmon an orbital shaker at 35° C. Sedimentary fungal filaments appeared inabout 36 hrs growing.

Ph-CA1 and Ph-CA2 were added at the start of the culture. The sameamount of borate stock solution (50 mM borate, PH9.0), Ph-SA(pheromonicin constructed by colicin Ia and staphylococcal pheromoneAgrD1)(10 ug/ml) and several antibiotics preparations (2 ug/mlamphotericin B, 6 ug/ml fluconazole) were used as controls. All assayswere expressed in turbidometric absorbance units measured at 595 nm andpictures of the filament sedimentation at the bottom of the flask weretaken.

Fluconazole and Ph-SA had no effect on the growth of C. albicanscompared to untreated controls. In contrast, 10 ug/ml Ph-CA1 completelyinhibited C. albicans growth, as did 2 ug/ml amphotericin B. 10 ug/mlPh-CA2 had about 30% of the inhibition effect as the Ph-CA1. Consideringthe difference in molecular weight between Ph-CA1 (70 kDa) andamphotericin B (about 0.9 kDa), the inhibitory effect of Ph-CA 1 againstC. albicans was approximately ten times greater, on a molar basis, thanthat of amphotericin B (see FIG. 4). The spores and filaments of 2 ultreated medium were dripped on a slide and observed under microscope. Incomparison with control and other treatments, spores were scarcelyobserved in the amphotericin B and Ph-CA1 (see FIG. 5).

FIG. 6 shows that after 24 hrs of incubation with Ph-CA1 (10 ug/ml),cell membrane of most C. albicans cells (stained by FITC as green in thepresence of propidium iodide) was damaged thus the propidium iodideentered into the cell to stain cells red.

KungMing mice, half male and half female, weighing 18-22 g were injectedintraperitoneally with 0.5 ml of C. albicans (ATCC 10231), 10⁸ CFU/ml.One hour after C. albicans injection, mice were injectedintraperitoneally with 0.9% saline (A) alone as control (n=10) (C), orwith amphotericin B (n=10, 1 ug/gm/day) (B), or with Ph-CA1 (n=10, 5ug/gm/day) (A) daily for 14 days. The number of surviving animals wasdetermined every 24 hours (FIG. 11).

KungMing mice, half male and half female, weighing 18-22 g were injectedintraperitoneally with 0.7 ml of C. albicans (ATCC 10231), 10⁸ CFU/ml.One hour after C. albicans injection, mice were injected in the tailvein with 0.9% saline alone as control (n=10) (C), or with amphotericinB (n=10, 1 ug/gm) (B), or with Ph-CA1 (n=10, 5 ug/gm) (A). The mice werethen injected intraperitoneally with 0.9% saline alone, or withamphotericin B (n=10, 1 ug/gm), or with Ph-CA1 (n=10, 5 ug/gm) each day.The number of surviving animals was determined every 24 hours (FIG. 12).Considering the difference in molecular weight between Ph-CA1 (70 kDa)and amphotericin B (about 0.9 kDa), the in vivo antifungal activity ofPh-CA1 against systemic candidiasis was at least twenty times greater,on a molar basis, than that of amphotericin B.

KungMing mice (n=10), half male and half female, weighing 18-22 g wereinjected intraperitoneally with Ph-CA1 (200 ug/mouse/day) for 20 days.The bodyweight of all mice was increased. There was no microscopicevidence of necrosis or inflammation in the livers, kidneys or spleensof mice (FIG. 13).

A 300 m² rice field (seed, gangyou 725) with Magnaporthe griseainfection was randomly divided as three zones. The middle 100 m² areawas treated with water spraying twice as control, the left 100 m² areawas treated with tricyclazole spraying twice (0.5 mg/ml and 1 mg/ml) andthe right 100 m² area was treated with Ph-CA1 spraying twice (1 ug/mland 2 ug/ml) at the tillering stage. The time interval between twosprayings was 7 days. Each 200 leaves were randomly examined in controland treatment areas to determine the protecting efficacy of Ph-CA1. Thedata are depicted below in Table I.

TABLE 1 Examining Grades of impaired leaves Incident Infected Protectingdate 0 1 3 5 7 9 rate index efficacy One day 152 27 14 6 1 24 5.88Before Treatment After 89 57 30 7 7 8 55.5 17.38 Treatment Seven 172 1012 6 14 4.22 75.83 days Tri- cyclazole Ph-CA1 67 12 13 8 16.5 5.05 70.94

Another 300 m² rice field (seed, gangyou 725) with Magnaporthe griseainfection was randomly divided as three zones. The middle 100 m² areawas treated with water spraying once as control, the left 100 m² areawas treated tricyclazole spraying once (1 mg/ml) and the right 100 m²area was treated with Ph-CA 1 spraying once (2 ug/ml) at the head stage.About 200 ears were randomly examined in control and treatment areas todetermine the protecting efficacy of Ph-CA1. The data are depicted belowin Table II.

TABLE II Grades of impaired ears Impaired Infected Damage 0 1 3 5 7 9ears rate index rate Control 178 33 22 11 2 2 28.6% 8.33  4.2% Tri- 18416 9 2 2 0 13.62%  3.5 1.63% cyclazole Ph-CA1 218 19 5 0 0 0 9.92% 1.560.53%

In both of the above in vivo protecting assays, the concentration ofPh-CA1 used was approximately 500 times smaller than that oftricyclazole. On a molar basis, the protecting effects of Ph-CA1 werethree hundred times greater than that of tricyclazole. With these twofactors taken together, the total effects of Ph-CA1 against rice blastdisease was approximately 10⁴ to 10⁵ times greater than that oftricyclazole.

One skilled in the'art will appreciate that, for this and otherprocesses and methods disclosed herein, the functions performed in theprocesses and methods may be implemented in differing order.Furthermore, the outlined steps and operations are only provided asexamples, and some of the steps and operations may be optional, combinedinto fewer steps and operations, or expanded into additional steps andoperations without detracting from the essence of the disclosedembodiments.

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the disclosure, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims.

The present disclosure is to be limited only by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. It is to be understood that this disclosure is notlimited. to particular methods, reagents, compounds compositions orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” oran (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, agroup having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims. All references recited herein are incorporated hereinby specific reference in their entirety.

The invention claimed is:
 1. A recombinant nucleic acid molecule comprising: a polynucleotide sequence encoding an antifungal polypeptide, wherein the antifungal polypeptide comprises: a fungal pheromone targeting agent; and a channel-forming domain of colicin Ia linked to the fungal pheromone targeting agent, and wherein the channel-forming domain comprises the amino acid sequence of SEQ ID NO:
 8. 2. The recombinant nucleic acid molecule of claim 1, wherein said pheromone is from a pathogenic fungus selected from the group consisting of Candidas, Aspergillus, Magnaporthes and Fusarium.
 3. The recombinant nucleic acid molecule of claim 2, wherein said Candidas, Aspergillus, Magnaporthes and Fusarium are selected from the group consisting of Candida albicans, Candida tropicalis, Candida parapsilosis , Candida krusei, Candida dubliniensis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Magnaporthe grisea and Fusarium moniliforme.
 4. The recombinant nucleic acid molecule of claim 1, wherein the colicin Ia is linked to the C-terminus of the fungal pheromone targeting agent.
 5. The recombinant nucleic acid molecule of claim 1, wherein the channel-forming domain of colicin Ia is linked to the N-terminus of the fungal pheromone targeting agent.
 6. The recombinant nucleic acid molecule of claim 1, wherein said antifungal polypeptide has a molecular weight of about 26,000 to about 70,000 daltons.
 7. A recombinant nucleic acid molecule comprising: a nucleic acid sequence encoding an antifungal polypeptide, wherein the antifungal polypeptide comprises: a Candida albicans alpha-mating pheromone; and a channel forming domain of colicin Ia linked to the Candida albicans alpha-mating pheromone, and wherein the channel-forming domain comprises the amino acid sequence of SEQ ID NO:
 8. 8. The recombinant nucleic acid molecule of claim 7, wherein said Candida albicans alpha-mating pheromone comprises the amino acid sequence of SEQ ID NO:
 3. 9. The recombinant nucleic acid molecule of claim 7, comprising the nucleic acid sequence of SEQ ID NO:
 2. 10. A recombinant nucleic acid molecule comprising: a nucleic acid sequence encoding an antifungal polypeptide, wherein the antifungal polypeptide comprises the amino acid sequence of SEQ ID NO: 5, and wherein the antifungal polypeptide has a fungal pheromone targeting agent, and a channel-forming domain of colicin Ia linked to the fungal pheromone targeting agent.
 11. The recombinant nucleic acid molecule of claim 10, comprising the nucleic acid sequence of SEQ ID NO:
 4. 12. A vector comprising the recombinant nucleic acid molecule of claim
 1. 13. A vector comprising the recombinant nucleic acid molecule of claim
 7. 14. A vector comprising the recombinant nucleic acid molecule of claim
 10. 15. A host cell comprising the vector of claim
 12. 16. A host cell comprising the vector of claim
 13. 17. A host cell comprising the vector of claim
 14. 18. The recombinant nucleic acid molecule of claim 1, comprising: the fungal pheromone targeting agent that comprises the amino acid sequence of SEQ ID NO:
 3. 19. The recombinant nucleic acid molecule of claim 1, wherein the polynucleotide sequence comprises the nucleotides of SEQ ID NO:
 2. 